In this research program, the non-linear optical methods of two-photon excitation (TPE) fluorescence and second harmonic generation (SHG) will be combined with near-field scanning optical microscopy (NSOM) to study aspects of membrane physiology. Due to the enhanced non-linear properties of available membrane staining dyes and decreased far-field background contributions, these approaches will provide increased contrast over existing linear excitation methods while maintaining sub- optical resolution. Chemical composition and physical properties are important in cell function but optical microscopy cannot provide the necessary resolution to uncover these details. At he local molecular level, properties such as membrane potential, elasticity, or hydrophilicity, will vary and these variations may be important in cell function. The NSOM apparatus will be used to correlate such properties to local chemical composition, e.g. cholesterol to lipid ratio, in living cells. Fluorescence lifetime measurements and time-dependent fluorescence anisotropy measurements will be used to probe the local environment around fluorophores. Second harmonic generation arises from an induced polarization rather than absorption and potential sensitive dyes will be used to generation good contrast without appreciable photobleaching. SHG combined with polarization analysis will be used study the orientation of fluorophores in membranes. SHG will also be used as an alternative method to measure membrane potential. The initial experiments will be on model cell line, e.g. 3T3 fibroblast and N1E-115 undifferentiated and differentiated neuroblastoma cells but once operational, the apparatus will be general purpose high resolution tool for live cell imaging. A key aspect of this proposal will be constructing cavity dumped titanium sapphire oscillator to provide higher pulse energy so that high signal levels can be obtained for the SHG experiments.